Currently, there is no good diagnostic test for typhoid fever (or carriage) in typhoid endemic areas of the world. This deficiency complicates the targeted administration of appropriate antimicrobials, and is a major impediment to the more widespread endorsement of typhoid control and vaccine programs. In this two-stage R21 (exploration)/R33 (expansion and development) proposal, we build upon an ongoing international collaborative effort to support the following specific aims and milestones: R21: years 1 and 2: innovative hypothesis-driven projects 1. Evaluation of anti-S. typhi IgA ALS fluid detection system as a typhoid diagnostic. 2. Development of urine ELISA or dipstick assay. 3. Screen asymptomatic carrier serum for antibodies targeting S. typhi antigens expressed in vivo and unique to the carrier state. Main milestones by the end of year 2 (R21), we propose (1) to have developed at least one prototype approach (either ALS-based or urine-based) to use in subsequent evaluations in Dhaka, Bangladesh, and (2) to have screened carrier blood to assess if a unique signal is present. R33: years 3-5: additional innovative exploratory research and expanded development of diagnostics 4. Field (endemic zone) test prototype assays in Dhaka, Bangladesh (ALS and/or urine device[s]) from R21 phase, if promising. 5. Advance carrier diagnostic, if promising. If not promising: 6. Further explore the use of a microfluidic mannose binding lectin capture system to increase recovery of S. typhi in the blood of acutely infected patients. 7. Explore interferon-gamma based detection assay (IGA) approach for typhoid. 8. Explore the ability to diagnostically detect S. typhi mRNA/cDNA (as opposed to gDNA) in the blood of infected humans with typhoid, to improve sensitivity. Main deliverable by the end of year 5 (R33): development of diagnostic approach(es) that is/are more sensitive and specific than current assays in an endemic zone (our initial goal will be >90% sensitivity; >90% specificity; a significant improvement over assays currently available/in use in endemic zones). PUBLIC HEALTH RELEVANCE: Typhoid fever affects over 20 million individuals per year, killing 200,000. There are good typhoid vaccines, but no good diagnostic test to know when to use them. Development of a good diagnostic test would facilitate use of currently available typhoid vaccines, assist in targeting use of appropriate antimicrobial agents, and would improve public health, potentially saving tens of thousands of lives per year.